Expression Analyses Revealed Thymic Stromal Co-Transporter/Slc46A2 Is in Stem Cell Populations and Is a Putative Tumor Suppressor

By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified TSCOT expression in thymic tissue- and cell type- specific fashion and is also expressed in some other epithelial tissues including skin and lung. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the organogenesis. TSCOT expression was detected in all thymic epithelial cells (TECs), but not in the CD31⁺ endothelial cell lineage in fetal thymus. In addition, ABC transporter-dependent side population and Sca-1⁺ fetal TEC populations both contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By searching variations in the expression levels, TSCOT is positively associated with Grhl3 and Irf6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung is diminished in lung cancers, suggesting TSCOT may be involved in the suppression of lung tumor development. Based on these results, a model for TEC differentiation from the stem cells was proposed in context of multiple epithelial organ formation.     

TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.     

Epithelial cell-specific laminin 5 is required for survival of early thymocytes

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.     

Expression of PAC1 receptor in rat thymus after irradiation

In the present work, PAC1-R (G-protein-coupled receptor specific for PACAP) was detected on cells in the normal thymus. Immunohistochemically PAC1-R was expressed strongly in stromal cells of the thymic medulla. Positive cells were also observed in the thymus of fetal and old adult rats. After 8 Gy irradiation to 9-week-old rats, PAC1-R expressions in the thymus decreased and almost recovered by day 21. The expression of PAC1-R mRNA was weak in the thymus and decreased further after irradiation. The expression almost recovered by day 28. Hip and hip/hop variants, which were not expressed in the normal thymus, were expressed in the thymus on days 3, 5 and 21 after irradiation. The expressions of IL-6 and IL-10 tended to increase initially after irradiation then decreased. Histologically, the thymic structures were destroyed on day 3 after irradiation and the thymus almost recovered by day 21. Thus PACAP is thought to be one of the important factors for cross-talk between cells involved in thymic regeneration.     

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.     

Development of thymic microenvironments in vitro is oxygen-dependent and requires permanent presence of T-cell progenitors.

Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.     

EVA regulates thymic stromal organisation and early thymocyte development

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.     

The myelopoietic inducing potential of mouse thymic stromal cells

The thymus has generally been considered as being solely involved in T cell maturation. In this study we have demonstrated that mouse thymic stroma can also support myelopoiesis. Bone marrow from mice treated with 5-fluorouracil was depleted of cells expressing Mac-1, CD4, and CD8 and incubated on lymphocyte-free monolayer cultures of adherent thymic stromal cells. After 7 days there was a marked increase in nonadherent cells, the majority of which were Mac-1+, FcR+, and HSA+. These proliferating bone marrow cells also expressed markers (MTS 17 and MTS 37) found on thymic stromal cells. Such cells were not found in thymic cultures alone, in bone marrow cultured alone, or on control adherent cell monolayers. Supernatants from the cultured thymic stroma, however, were able to induce these cell types in the bone marrow precursor population. Incubation of normal thymocytes with a monolayer of these in vitro cultivated Mac-1+, MTS 17+, MTS 37+ myeloid cells leads to selective phagocytosis of CD4+ CD8+ cells. Hence, this study demonstrates that the thymic adherent cells can induce myelopoiesis in bone marrow-derived precursor cells and provide a form of self-renewal for at least one population of thymic stromal cells. Furthermore, these induced cells are capable of selective phagocytosis of CD4+ CD8+ thymocytes and may provide one mechanism for the selective removal of such cells from the thymus.     

Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development

The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.     

Thymic T-cell tolerance of neuroendocrine functions: physiology and pathophysiology.

Intimate interactions between the two major systems of cell-to-cell communication, the neuroendocrine and immune systems, play a pivotal role in homeostasis and developmental biology. During phylogeny as well as during ontogeny, the molecular foundations of the neuroendocrine system emerge before the generation of diversity within the system of immune defenses. Before reacting against non-self infectious agents, the immune system has to be educated in order to tolerate the host molecular structure (self). The induction of self-tolerance is a multistep process that begins in the thymus during fetal ontogeny (central tolerance) and also involves anergizing mechanisms outside the thymus (peripheral tolerance). The thymus is the primary lymphoid organ implicated in the development of competent and self-tolerant T-cells. During ontogeny, T-cell progenitors originating from hemopoietic tissues (yolk sac, fetal liver, then bone marrow) enter the thymus and undergo a program of proliferation, T-cell receptor (TCR) gene rearrangement, maturation and selection. Intrathymic T-cell maturation proceeds through discrete stages that can be traced by analysis of their cluster differentiation (CD) surface antigens. It is well established that close interactions between thymocytes (pre-T-cells) and the thymic cellular environment are crucial both for T-cell development and for induction of central self-tolerance. Particular interest has focused on the ability of thymic stromal cells to synthesize polypeptides belonging to various neuroendocrine families. The thymic repertoire of neuroendocrine-related precursors recapitulates at the molecular level the dual role of the thymus in T-cell negative and positive selection. Thymic precursors not only constitute a source of growth factors for cryptocrine signaling between thymic stromal cells and pre-T-cells, but are also processed in a way that leads to the presentation of self-antigens by (or in association with) thymic major histocompatibility complex (MHC) proteins. Thymic neuroendocrine self-antigens usually correspond to peptide sequences highly conserved during the evolution of their corresponding family. The thymic presentation of some neuroendocrine self-antigens does not seem to be restricted by MHC alleles. Through the presentation of neuroendocrine self-antigens by thymic MHC proteins, the T-cell system might be educated to tolerate main hormone families. More and more recent experiments support the concept that a defect in thymic tolerogenic function is implicated as an important factor in the pathophysiology of autoimmunity.     

Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus.

A hamster monoclonal antibody (MAb), designated 8.1.1, was raised against murine thymic stromal cell lines and was found to react with cell surface molecules expressed by a morphologically distinct population of epithelial cells of the murine thymus comprising the subcapsular environment, cells investing vascular structures throughout the thymus, and some of the cellular elements in the medulla. The epithelial nature of the labeled cells was confirmed with immunoelectron microscopy. Reactivity with MAb 8.1.1 was associated with thymic epithelial cells in contact with basal laminae. Ontological studies of thymic tissue demonstrated that the epitope recognized by this MAb was expressed before Day 14 of gestation, although the restricted subcapsular and medullar expression of 8.1.1 was not apparent until sometime after birth. MAb 8.1.1 also reacted with a number of extra-thymic tissues, including lamina propria of gut, glomeruli and tubules in the kidney, mesothelia covering a number of organs, and the dermis and epidermis of skin. Within the epidermis, reactivity of MAb 8.1.1 was largely restricted to basal epithelial cells. Immunochemical analysis of 8.1.1 reactivity with detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that the epitope recognized by this MAb was associated with a glycoprotein bearing terminal N-acetylglucosamine residues and possessing an Mr of approximately 36-38 KD under reducing or non-reducing conditions.     

Human thymic epithelial cells are frequently transformed by retroviral vectors encoding simian virus 40.

The thymic microenvironment contains a mixture of phenotypically distinct epithelial cells of varied functions, some of which are unknown. In an attempt to understand their relevance to T cell differentiation in the thymus, human thymic epithelial cell clones from both fetal (SM3-SM5) and postnatal (SM6) thymus were produced by using a defective recombinant retroviral vector encoding the simian virus 40 large T antigen and the neomycin resistance gene. The presence of keratins 8 and 18, desmosomes, and tonofilaments confirmed the epithelial origin of the cell strains. The cells expressed Thy-1 and HLA-Class I at high levels, showed weak-expression antigens defined by TE3B and A2B5, and low to negligible levels of the MR19-defined molecule. When compared with the phenotype of thymic epithelial cells in situ, the cell strains appear to be derived from neuroendocrine components in the outer cortical region of the human thymus. The use of retroviral vectors to transform human thymic epithelium was considerably more efficient than transfection with a plasmid carrying the origin of replication-defective SV40 large T gene. In the latter case, only two cell strains with subcapsular epithelial phenotypes were derived from fetal thymus. With the retroviral vectors, epithelial cell strains could, for the first time, be generated from human postnatal thymus as well as from fetal thymus.     

Thymic hormones in radiation-induced immunodeficiency. I. Induction of mature interleukin 1 responsive cell in the thymus by thymosin fraction 5

The restorative effect of thymosin fraction 5 (TF5) on the thymus of gamma-irradiated mice was examined. Four different mouse strains were used in this study since earlier work determined that the degree of response to TF5 is strain dependent. The responsiveness to comitogenic effect of interleukin 1 (IL-1) was used to measure the rate of recovery of immunocompetent cells in the thymus, since only more mature PNA-, Lyt-1+-2- medullary cells respond to this monokine. Contrary to several earlier reports that radioresistant cells repopulating the thymus within the first 10 days after irradiation are mature, corticosteroid resistant, immunocompetent cells, the thymic cells from irradiated mice in all strains used had greatly reduced responses to IL-1. Daily intraperitoneal injections of TF5 increased significantly the responses of thymic cells to IL-1 in 10- to 13-weeks-old C57Bl/KsJ, C57Bl/6, C3H/HeJ, and DBA/1 mice. Older mice, 5 months or more in age, of DBA/1 strain did not respond to treatment with TF5. However, C3H/HeJ mice of the same age were highly responsive. In conclusion, (1) cells repopulating the thymus within 12 days after irradiation contain lower than normal fraction of mature IL-1 responsive cells, (2) thymic hormones increase the rate of recovery of immunocompetent cells in the thymus, and (3) the effect of thymic hormones is strain and age dependent.     

Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors

Interactions between T cell precursors and thymic stromal cells are essential during thymocyte development. However, the role of the thymus in initial commitment of lymphoid progenitors to the T lineage remains controversial, with data providing evidence for both extra- and intrathymic commitment mechanisms. In this context, it is clear that Notch1 is an important mediator during initiation of T cell development. Here we have analyzed the mechanisms regulating Notch activation in lymphoid precursors at extrathymic sites and in the thymus, including stages representing the first wave of embryonic thymus colonization on embryonic day 12 of gestation. We show that Notch activation in migrant lymphoid precursors requires entry into the thymic microenvironment where they are exposed to Notch ligands expressed by immature thymic epithelial cells. Moreover, continued Notch signaling in such precursors requires sustained interactions with Notch ligands. Collectively, these findings suggest a role for Notch in an intrathymic mechanism of T cell lineage commitment involving sustained interactions with Notch ligand bearing thymic epithelium.     

The effect of graded doses of fission neutrons or X rays on the stromal compartment of the thymus in mice

The effect of irradiation on the supportive role of the thymic stroma in T cell differentiation was investigated in a transplantation model using athymic nude mice and transplanted irradiated thymuses. In this model, neonatal CBA/H mice were exposed to graded doses of whole-body irradiation with fast fission neutrons of 1 MeV mean energy or 300 kVp X rays. The doses used varied from 2.75 up to 6.88 Gy fission neutrons and from 6.00 up to 15.00 Gy X rays at center-line dose rates of 0.10 and 0.30 Gy/min, respectively. Subsequently, the thymus was excised and a thymus lobe was transplanted under the kidney capsule of H-2 compatible nude mice. One and two months after transplantation, the T cell composition of the thymic transplant was investigated using immunohistology with monoclonal antibodies directed to the cell surface differentiation antigens Thy-1, Lyt-1, Lyt-2, MT-4, and T-200. Furthermore, the stromal cell composition of the thymic transplant was investigated with monoclonal antibodies directed to MHC antigens and with monoclonal antibodies defining different subsets of thymic stromal cells. To investigate the reconstitution capacity of the thymic transplant, the peripheral T cell number was measured using flow cytofluorometric analysis of nude spleen cells with the monoclonal antibodies anti-Thy-1, anti-Lyt-2, and anti-MT-4. The results of this investigation show that a neonatal thymus grafted in a nude mouse has a similar stromal and T cell composition as that of a normal thymus in situ. In addition, grafting of such a thymus results in a significant increase of the peripheral T cell number. Irradiation of the graft prior to transplantation has no effects on the stromal and T cell composition but the graft size decreases. This reduction of size shows a linear dose-response curve after neutron irradiation. The X-ray curve is linear for doses in excess of 6.00 Gy. The RBE for fission neutrons for the reduction of the relative thymic graft size to 10% was equal to 2.1. Furthermore, the peripheral T cell number decreases with increasing doses of irradiation given to the graft prior to transplantation. The present data indicate that the regenerative potential of thymic stromal cells is radiosensitive and is characterized by D0 values equal to 2.45 and 3.68 Gy for neutrons and X rays, respectively. In contrast, the ability of the thymic stromal cells to support T cell maturation is highly radioresistant.